We propose to study the regulation of mitochondrial function, in particular oxidative phosphorylation and proton-coupled processes by the physical properties of the mitochondrial inner membrane. As a working hypothesis, we suggest that, in addition to the chemiosmotic, bulk-phase proton transfer, a direct proton transfer between the ATPase and the electron transport system may also occur. This direct coupling is expected to be dependent on membrane fluidity, protein aggregation and mobility. This may also lead to membrane domains in which protons do not equilibrate with the medium. The hypothesis would be tested by comparing the correlations between the rate and efficiency of oxidative phosphorylation, and the proton electrochemical gradient, to the correlations between oxidative phosphorylation and membrane properties such as fluidity, phase structure and protein aggregation and mobility. In particular the effects on these correlations of membrane perturbing agents such as general anesthetics, local anaesthetics, lipophilic ions and other non-classical uncouplers or energy transfer inhibitors will be studied in detail. Also we shall investigate whether the rapid effect of thyroid hormones on oxidative phosphorylation and of glucagon is related to changes in membrane interaction or the proton electrochemical potential. The suggested experiments require further development and improvement in various techniques. We shall attempt further improvement and refinement in the measurement of the proton electrochemical potential and particularly membrane potential. A method for the measurement of surface charge and local charge, which is based on Tb+3 fluorescence, and is free from interference from the membrane potential, would be further developed and applied in these studies. Fluorescent pH indicators for membrane lipid and membrane protein will be tested. The use of phosphorescent "triplet" probes for measurement of protein mobility and aggregation will be adopted for these studies. These studies should contribute to a better understanding of the mechanism of oxidative phosphorylation and its regulation in health and disease.